Development and standardization of a protocol for sperm cryopreservation of two important commercial oyster species

Dissertação de mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Aquaculture activities have a huge contribution for the world food production and their development is extremely necessary to answer to the lack of resources, especially to the demand for seafood. Bivalve production, especially Crassostrea angulata (Portuguese oyster) has been practiced from long ago, and although its production suffered several constraints, in recent years it has been increasing the interests in recovering production and in preserving nature populations. In this sense, new research needs to guarantee an efficient and economically viable production, contributing to a relatively new environmental concern: wild population restoration. Nowadays, pure wild populations of Crassostrea angulata are rare to find due to multiple factors that affected this oyster industry. Cryopreservation technology could promote alternative techniques to contribute for the resource management efficiency of the Portuguese oyster and associated economic activity. In this sense, standardization of procedures is important for Crassostrea genus. At the present there are no cryopreservation reports on Crassostrea angulata sperm, and therefore, one of the objectives of this work is to design a cryopreservation protocol for this species, testing the more adequate cryoprotectant solution, its ideal concentration, different freezing rates and types of containers. In parallel, this stablished protocol was applied in Crassostrea gigas and compared to other previously published for this species. Analysis of motility, viability, agglutination and fertilizations were used as guides for the establishment of the protocol in C. angulata. Moreover, ATP content, DNA fragmentation and lipid peroxidation were done in order to standardize the same protocol for both species. Movement analysis were assessed by CASA system, viability through common staining techniques and flow cytometer, agglutination was quantified according to the scale developed by Dong et al., (2007), ATP content determined by bioluminescence, Comet assay was performed to quantify the DNA fragmentation and lipid peroxidation determined spectrophotometrically by measuring the absorbance of the malondialdehyde (MDA). Significant differences were observed (p<0.05) for lipid peroxidation and fertilization trials whereas ATP content and fragmentation of DNA of the cryopreserved samples did not differ significantly from the control. In C. gigas, the same analysis were performed and did not reveal post-thaw quality differences in the samples cryopreserved with 10% DMSO. The established protocol revealed to be effective and with a low degree of cellular damage on C. angulata sperm and, at the same time, viable to apply in other species, such as Crassostrea gigas.

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